Comparison of Hydroperoxide Initiator Requirements for the Cyclooxygenase Activities of Prostaglandin H Synthase-1 and −2

Abstract

Two isoforms of prostaglandin H synthase have been described: isoform-1 (PGHS-1), which is ascribed a role in basal or housekeeping prostaglandin synthesis; and isoform-2 (PGHS-2), which has been found to be strongly inducible in many tissues and has been associated with inflammatory processes. Recent observations have indicated that cyclooxygenase catalysis by the two isoforms can be differentially regulated when both are present simultaneously (Reddy, S. T., and Herschman, H. R. To compare the levels of hydroperoxide required for cyclooxygenase initiation in the two PGHS isoforms, we have examined the ability of a hydroperoxide scavenger, glutathione peroxidase, to suppress the cyclooxygenase activity of purified preparations of human PGHS-2, ovine PGHS-2, and ovine PGHS-1. Half-maximal prostaglandin synthetic activity was found to require a much lower hydroperoxide level with human PGHS-2 (2.3 nM) and ovine PGHS-2 (2.2 nM) than with ovine PGHS-1 (21 nM). Similar results were obtained when cyclooxygenase activity was monitored by chromatographic analyses of radiolabeled arachidonate metabolites or with oxygen electrode measurements. Mixing four parts of ovine PGHS-1 with one part of human PGHS-2 did not markedly change the sensitivity of the overall cyclooxygenase activity to inhibition by glutathione peroxidase, indicating that the PGHS-1 activity was not easily initiated by PGHS-2 activity in the same vessel. Effective catalysis by PGHS-2 can thus proceed at hydroperoxide levels too low to sustain appreciable catalysis by PGHS-1. This difference in catalytic characteristics provides a biochemical mechanism for differential control of prostaglandin synthesis by the two PGHS isoforms, even when both are present in the same intracellular compartment.

Role of Asn-382 and Thr-383 in Activation and Inactivation of Human Prostaglandin H Synthase Cyclooxygenase Catalysis

“…In each case, the major immunoreactive species was a multiplet with an average M r of about 73 kDa (data not shown), similar to previous results (12), thus confirming expression of the full-length recombinant proteins in detergent-extractable form.…”

“…Assessment of Cyclooxygenase Sensitivity to Suppression by Glutathione Peroxidase-The cyclooxygenase activity of a fixed amount (typically about 30 units) of wild-type or mutant PGHS-2 protein or purified ovine PGHS-1 was assayed in the presence of varying amounts of cGPx following the procedure described previously (12). The reaction mixture contained 0.5 mM glutathione in addition to the other components of the standard cyclooxygenase assay described above.…”

“…Tween 20 was purchased from Pierce Chemical Co. Recombinant wild-type and mutant PGHS-2 were expressed in Sf9 cells using a baculovirus vector as described below and reconstituted with excess heme before use (12). PGHS-1 was purified to homogeneity from ovine seminal vesicles as the apoenzyme (14) and reconstituted with excess heme before use.…”

“…In intact fibroblasts, PGHS-1 cyclooxygenase catalysis was found to accelerate more slowly than PGHS-2 cyclooxygenase catalysis, and addition of peroxide-generating reagents increased cyclooxygenase catalysis (9), indicating that the cellular peroxide level can be a key element in differential control of cyclooxygenase catalysis by the two isoforms in vivo. A variety of glutathione peroxidase isozymes, including cytosolic glutathione peroxidase (cGPx), contribute to controlling cellular peroxide levels (10), and titration with cGPx has proven a useful biomimetic approach for quantitative characterization of the regulation of cyclooxygenase activity by peroxide (11,12). PGHS-1 cyclooxygenase activity is much more readily suppressed by cGPx than is PGHS-2 cyclooxygenase (12, 13), consistent with the observation of active PGHS-2 cyclooxygenase catalysis in many cells where the PGHS-1 cyclooxygenase remains latent (6).…”

Acetylation of prostaglandin H2 synthases by aspirin is inhibited by redox cycling of the peroxidase

“…Oxidation of Tyr385 by peroxidase catalysis is more efficient for PGHS-2 than for PGHS-1 [35,36], and therefore it is conceivable that hydroperoxide concentrations below the threshold for oxidation of Tyr385 in PGHS-I could engender Tyr385 formation in PGHS-2. Whether this could produce some isoform selectivity for aspirin at low concentrations of hydroperoxide deserves investigation.…”